Journal of Equine Veterinary Science 98 (2021) 103372

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Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Original Research

Evaluation of Equine Infectious Anemia Virus by the Indirect Enzyme-

linked Immunosorbent Assay EIA-LAB as Screening Tools in Mexico
Maria Carla Rodríguez Domínguez a, Roberto Montes-de-Oca-Jime
nez b, *,

zquez Chagoyan b, Alberto Barbabosa Pliego b,
Juan Carlos Va

lez a,
Jorge Antonio Varela Guerrero b, Laura Ileana Coroas Gonza
Salvador Lagunas Bernabe
b, c

a
Viral and Bacterial Vaccines Producer Enterprise, LABIOFAM, Havana, Cuba
b
Faculty of Veterinary Medicine and Zootechnics, Autonomy University of the State of Mexico, Toluca, Mexico
c
Research and Advanced Studies in Animal Health Center, Animal Health Diagnosis Department, Serology Laboratory, Toluca, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Equine infectious anemia is a worldwide distributed disease that affects the Equide family. Commercial
Received 9 December 2020 effective vaccine is not available, for that reason control of the disease depends on diagnostic tools. To
Received in revised form improve the efficiency of the diagnostic program in Cuba, LABIOFAM Group, developed an indirect
27 December 2020
enzyme-linked immunosorbent assay (ELISA), ELISA kit, to complement the diagnostic system that
Accepted 29 December 2020
currently uses the agar gel immunodiffusion (AGID) kit. The ELISA AIE-LAB Kit was evaluated in a
Available online 06 January 2021
Mexican context, compared with the gold standard test Agar gel immunodiffusion, AGID AIE-LABIOFAM,
and commercial AGID kit. The analytical sensitivity was determined using serial dilutions twofold of the
Keywords:
Equine
positive control serum to establish the range of detected antibodies in relation to the cutoff value of the
EIAV plate (OD 0.300). A precision study was carried out to evaluate repeatability, intermediate precision, and
ELISA reproducibility by estimating the standard deviation and coefficient of variation. The precision results
AGID were satisfactory and the values of the coefficient of variation were considered adequate to guarantee an
Diagnostic excellent consistency of the ELISA AIE-LAB. The diagnostic performance of the ELISA AIE-LAB involved
the evaluation of specificity, sensitivity, and concordance in comparison with both AGID tests. The
diagnostic sensitivity was 100% and the specificity 97.6%, with a very good degree of concordance
(Kappa ¼ 0.9). The results suggest that the ELISA AIE-LAB test could be used in Mexico as a diagnostic
system for the detection of specific antibodies against the equine infectious anemia virus, as per current
international norms.
© 2021 Elsevier Inc. All rights reserved.

1. Introduction significant economic importance for horse industry. Currently,

Equine infectious anemia virus (EIAV) is a member of Retro-
viridae family, genus Lentivirus, recognized as a pathogen with
Animal welfare/ethical statement: The research was performed in accordance with
the ethical standard laid down in the 1996 declaration of Helsinki and its later
amendments.
Conflict of interest statement: The authors declare that they have no conflict of
interest.
* Corresponding author at: Roberto Montes-de-Oca-Jime
nez, Faculty of Veteri-
nary Medicine and Zootechnics, Autonomy University of the State of Mexico, Toluca,
Mexico.
E-mail address: romojimenez@yahoo.com (R. Montes-de-Oca-Jime
nez).
there is no vaccine or treatment for EIA, and the control of the
disease depends on diagnostic tools’ efficacy [1].
Agar gel immunodiffusion (AGID) also known as Coggins test [2]
is an effective tool for detection of specific EIAV antibodies based on
the immunoreaction of antigen and samples’ antibodies. As per the
World Organization for Animal Health (OIE), AGID is considered as
the confirmatory standard test for the diagnosis of the disease [3].
Results’ interpretation is conducted by visual reading of a precipi-
tation line that can be highly subjective generating many samples
misinterpreted by inexperienced laboratory personnel [4].
During the last few years, the detection of EIA antibodies by
enzyme-linked immunosorbent assay (ELISA) has been applied in
some countries using different designs and antigens [5e7].

https://doi.org/10.1016/j.jevs.2021.103372
0737-0806/© 2021 Elsevier Inc. All rights reserved.

zquez Chagoyan et al.
M.C. Rodríguez Domínguez, R. Montes-de-Oca-Jim
enez, J.C. Va
Validation assays have pointed excellent agreement between these
ELISAs and the gold standard test [6,7]. In addition, scientific re-
ports have indicated that ELISAs are more sensitive technique than
AGID, with a higher sample processing capacity [8e10].
Most AGID and ELISA diagnostic kits allow the detection of p26
antibodies, but in recent years, other EIAV proteins have been used
as antigens for enhancing the sensitive of tests. Glycoprotein gp45
is an immunogenic viral envelope protein that mediates virus-host
membrane fusion and has been used as antigen in different ELISA
tests [11e13].
Routine diagnosis process established by Cuban sero-
surveillance program for EIA use the antigen-antibody AGID-AIE
Kit produced by LABIOFAM. Recently, this laboratory also has
developed an indirect ELISA AIE-LAB test using gp45 synthetic
peptide which has the advantage of allowing efficient and accurate
processing of a lot of serum samples’ number within a relatively
short period and of providing an objective interpretation by the use
of a spectrophotometer. Previews validation studies using equine
serum from different regions of Cuba have indicated a good
agreement between these ELISA and AGID-AIE LABIOFAM diag-
nostic kit. However, ELISA AIE-LAB has not been validated in other
conditions outside Cuba. One of the evidences about value and
efficacy of a diagnostic test is that capacity of giving successful
results in other laboratories under different conditions and coun-
tries [14].
Mexico has a worldwide consolidated equine industry, with a
population that was estimated on 6 385 102 animals, in accordance
with data provided by FAOSTAT for 2018 [15]. Equine are valuable
for agriculture, sport, and meat production [16,17] and for all these
activities is important count with a certainly diagnostic system that
establish that animals are free of EIAV. Information of the virus in
Mexico is limited [18]. The commercially available cELISA (IDEXX)
designed to detect antibodies to p26 that has been approved for EIA
diagnosis in several countries. Recently a study of serological
diagnosis using a commercial cELISA (IDEXX, USA) indicated the
presence of EIAV in 37.7% of a 56 population of work equines at the
municipality of Veracruz, Mexico [19].
For all that reason, the aim of this study was to evaluate the
performance of ELISA AIE-LAB in a Mexican context, using AGID-
AIE Kit (LABIOFAM, Cuba) and DyaSystems EIA-AGID test kit
(IDEXX, USA) as quality control diagnostic kits. For the first time, we
presented ELISA AIE-LAB results, a designed and produced kit by
Cuba industry, used for the diagnostic of EIAV with Mexican
samples.
2. Material and Methods
2.1. Serum Samples
A panel of 96 individual horse serum samples (86 negative and
10 positive) collected from State of Mexico, donned by the Research
and Advanced Studies in Animal Health Center (CIESA) and Na-
tional Center of Animal Health Diagnostic Services (CENASA) was
used in the study for the evaluation of ELISA AIE-LAB.
2.2. Agar Gel Immunodiffusion, AGID Test
AGID-AIE Kit (LABIOFAM, Cuba) and DyaSystems EIA-AGID test
kit (IDEXX, USA) were used following the manufacturer’s recom-
mendations and also OIE manual indications for EIA diagnosis [3].
AGID tests were performed, in a 15  90 mm diameter petri dish
with 15 mL of 1% Noble agar. After hardened, agar was perforated
with a mold that originates a central and six peripheral wells. The
measure of these was 5.3 mm in diameter and 2.4 mm of distance
between wells. Antigen p26 protein (24 mL) was placed in the
2
Journal of Equine Veterinary Science 98 (2021) 103372
central well and positive control sera interleaved with diagnostic
target serum samples (24 mL) were placed in peripheral wells. The
AGID test results were interpreted either as positive by visual
reading of precipitation line curvature or negative by the absence of
the line, after 48 hourse72 hours of incubation at room tempera-
ture (20 Ce25 C). The AGID tests were considered valid only if the
negative and positive controls included on each test plate yielded
the expected results. Samples were retested when interpretation of
the results was doubtful.
2.3. Enzyme-linked Immunosorbent Assay
ELISA test was carried out following the manufacturer in-
structions and OIE manual recommendations [3]. Serum samples
(5 mL) and buffer dilution (95 mL) were added in microtitration
plates provided by the manufacturer and incubated at 37 C for
1 hour. Then six steps of washed with buffer (PBS-Tween 20) were
carried out and anti-equine IgG-conjugated peroxidase (100 mL/
well) was added. An incubation step was performed at 37 C for
1 hour and later plates were washed four times. Orthophenylene-
diamine tablet (Sigma-Aldrich, USA) in phosphate-citrate sub-
strate buffer (100 mL) was added per well and incubated at 37 C for
15 minutes. Finally, reactions were stopped with 100 mL of 2M
H2SO4, and absorbance was read at 492 nm in a microplate Spec-
traMax M5 reader (Molecular Devices, USA).
2.4. Data Analysis
Some general aspects were analyzing such as content avail-
ability of the liquid components, manual of instruction, and
organoleptic properties by visual examination. The analytical
sensitivity was evaluated using twofold serial dilutions of the
positive control serum from 1/5 to 1/1280, to determine the
maximum dilution where antibodies were detected considering the
cutoff value of the plate, optical density (OD) 0.300.
A precision study was performed to estimate the repeatability,
intermediate precision, and reproducibility. The repeatability was
determined using 12 replicates of positive and negative serum
control from the ELISA and a positive field serum. For the inter-
mediate precision estimation, the design was the same as the
repeatability assay but evaluated in three different days. The
reproducibility was assessed in two laboratories with different
equipment and personnel. The standard deviation and the intra-
assay and interassay variation coefficients (CV) were also calcu-
lated [20]. The diagnostic performance of ELISA AIE-LAB included
the evaluation of specificity, sensitivity, and concordance against
both AGID tests [21,22]. In addition, robustness of ELISA was
demonstrated by the evaluation of absorbance value using conju-
gate reagent after 24 hours at room temperature.
3. Results
ELISA AIE-LAB kit contains 10 components that were supplied in
enough quantity for the evaluation of 192 samples. Liquid compo-
nents showed good transparency without sediments in none of the
flasks. In addition, the color of these components did not change in
the flasks or in the microplate during the assays. The evaluation of
ELISA AIE-LAB robustness challenging the anti-IgG equine peroxi-
dase conjugate at room temperature for 24 hours did not affect its
functionality. Furthermore, changes of the physical appearance of
the sera samples (lipemic and hemolytic sera) did not influence in
the expected results.
ELISA AIE-LAB allowed the detection of antibodies from a 1/5
dilution of the positive control serum to a 1/160, which represents
the detection limit of the assay, maximum and minimum range of

zquez Chagoyan et al.
M.C. Rodríguez Domínguez, R. Montes-de-Oca-Jim
enez, J.C. Va
Fig. 1. ELISA AIE-LAB evaluation assays for performance of diagnostic kit in Mexico.
the OD values detectable with the kit. The controls’ sera results
were OD of positive control serum > 1.0 and OD of negative control
serum between 0.025e0.05 and (P/N)  6.0 (P: OD media of posi-
tive control and N: OD media of negative control).
The validation parameters evaluated were repeatability and
reproducibility based on standard deviation (SD) and CV estima-
tion. In addition, accuracy was estimate from concordance (Kappa
index), diagnostic sensitivity, and specificity with respect to those
of the AGID (Fig. 1). For the repeatability trials (intraplate vari-
ability), all CV values were less than 20% as expected for both
controls’ serums and a positive serum from CENASA, Mexico
(Table 1). The CV values confirmed the satisfactory compliment of
valid parameters of ELISA AIE-LAB in the repeatability assay. The
results of repeatability trials to determine the intermediate preci-
sion of the system are summarized (Table 2). The reproducibility
results of trials showed CV values of 0.24 for positive control serum,
11.72 for negative control serum, and 2.26 for positive serum from
SENASA, Mexico (Table 3). All CV values obtained were lower than
20%. The precision results (repeatability and reproducibility) were
satisfactory, and CV values were considered adequate to guarantee
excellent precision and consistency of the ELISA AIE-LAB (Fig. 1).
Correlation between ELISA AIE-LAB and AGID test results was
similar for both AGID techniques used as confirmatory control test.
Only two samples (sample 2 and 3) were identified as reactive
serums by ELISA AIE-LAB and were detected as negative by both
AGID test (Table 4) (Fig. 2).
The parameters of performing diagnosis for the ELISA in com-
parison with the AGID technique are showing in Table 5. The ELISA
Table 1
Repeatability study of the ELISA AIE-LAB (intraplate variability).
Positive Control Serum Negative Control Serum
X SD CV X SD
1.92 0.08 4.25 0.09 0.01
Abbreviations: X, mean value; SD, standard deviation; CV, coefficient of variation.
3
Journal of Equine Veterinary Science 98 (2021) 103372
AIE-LAB showed a 100% sensitivity and 97.6% specificity, with a
concordance 0.9, values considered adequate for the test.
4. Discussion
Equine infectious anemia virus causes a persistent infection
where animals remain viraemic carriers for life. Antibody response
usually persists and antibody-positive animals older than 6 months
are identified as infectious with the potential to transmit the virus
to other horses. Currently, there is no vaccine or effect treatment for
EIA, and that is why the control strategy consists in the detection
and segregation of infected animals [1,3]. The AGID or Coggins test
have been used since 1973 [23] as the official diagnosis test
established by OIE, also prescribed as mandatory test for interna-
tional movement of equine [14]. However, this test has a low
sensitivity which can originate false results making more difficult of
the control and eradication of EIA. Even though the specificity of
the AGID test is very high, its sensitivity is lower, and horses can
transmit EIAV even though their AGID tests were negative. In
addition, to develop a more rapid and sensitive diagnostic tools,
several laboratories have developed ELISA [7,10].
The ELISA AIE-LAB is an indirect heterogenic system designed
and produced in Cuba with the purpose of enhancing the efficiency
of the National Diagnostic Program of EIA. The ELISA EIA-LAB uses a
synthetic peptide from gp45 protein as antigen for detection of
EIAV antibodies presenting advantages over the use of natural an-
tigens because they increase the sensitivity and eliminates crossed
reactions [24]. The primary goal of this study was to evaluate the
Positive Serum
CV X SD CV
14.38 1.83 0.04 2.06

zquez Chagoyan et al.
M.C. Rodríguez Domínguez, R. Montes-de-Oca-Jim
enez, J.C. Va Journal of Equine Veterinary Science 98 (2021) 103372

Table 2 Table 4
Intermediate precision of ELISA AIE-LAB. Diagnosis Comparison of AGID EIA IDEXX Commercial System: AGID AIE-LABIOFAM
and ELISA AIE-LAB.
Days Positive Control Negative Control Positive Serum
Serum Serum ELISA AIE-LAB AGID IDEXX and AGID AIE-LABIOFAM

X SD CV X SD CV X SD CV Positive Negative Total

1 1.92 0.08 4.25 0.09 0.01 14.38 1.83 0.04 2.06 Positive 10 2 12
2 2.15 0.12 5.46 0.09 0.02 17.53 1.93 0.03 1.72 Negative 0 84 84
3 2.24 0.16 7.06 0.12 0.02 13.93 1.96 0.05 2.77 Total 10 86 96

Abbreviations: X, mean value; SD, standard deviation; CV, coefficient of variation.

ELISA AIE-LAB performance in CIESA laboratory of UAEM Univer-
sity. The components provided in the kit presented adequate
organoleptic characteristics (color and texture) in accordance with
establish by the manufacturer. The color present in positive and
negative serum controls facilitates identification of the reagents.
The design of color change of control serum at contact with the
reagent sample diluent allows easy visualization in the support of
the reaction. The chemical properties and functional characteristic
of the components were satisfactory to achieve established limit of
OD. The detection limit established by the manufacturer provided
an extensive range of dilutions for the positive serum that increase
the sensitivity of the technic. The anti-IgG equine-peroxidase
conjugate activity was not affected by change in temperature. All
these results were a sample of the robustness of the ELISA AIE-LAB,
which indicates the ability to maintain the same results in the face
of small changes that may occur during the test.
The repeatability is an indicator of the coincident results be-
tween the replica of the same sample in one or different trials. At
least a minimum of three samples representing analytic activity
within the operating range of the assay have to be used for the
repeatability evaluation. Reproducibility is the ability of a diag-
nostic test to provide consistent results, as a parameter of precision,
when the evaluation of aliquots of the same sample tested with a
kit in different laboratories, located in distinct regions or countries,
achieves the same results [14].
ELISA EIA-LAB showed a satisfactory result in repeatability trials
(intraplate variability) and reproducibility because all CV values
were less than 20%. The CV values of positive serum were less than
15%, however, CV of the negative serum had a different behavior
associated with low-OD values (<1.0). Small numerical variations in
OD of negative serums can produce higher CV values because the
calculation of CV (CV ¼ standard deviation/mean of OD) using a low
value of denominator (low mean OD) mathematically produce a
higher numerical result. These results are in agreement with
another ELISA kits in repeatability assay when CV values obtain had
been 2.8% [11] and 12.73% [25] for positive sera and less than 20%
for the negative serum [26e28]. Previously reported CV values of
reproducibility for positive samples have been between the range
of 3 and 15% [11,13] and CV values for negative samples even more
than 20% [11,25].
The comparison of ELISA EIA-LAB and AGID test originated two
discordant results, which have been expected because OD values
for these two serums were around the gray zone established by the
Table 3
Reproducibility results of ELISA AIE-LAB.
Laboratory Positive Control Negative Control
Serum Serum
X SD CV X SD CV
Lab 1 2.24 0.16 7.06 0.12 0.02 13.93
Lab 2 2.25 0.11 4.76 0.11 0.02 16.66
Abbreviations: X, mean value; SD, standard deviation; CV, coefficient of variation.
manufacturer, which indicates that these are weak serums and are
not visible in the precipitation line by AGID. Lower antibody con-
centration in a serum sample may originate negative AGID inter-
pretation, while are positive by ELISA [5,10,11]. In addition, positive
ELISA results may be due to the presence of gp45 antibodies, pro-
duced in the early stages of viral infection, which are not possible to
detect by AGID test because it only uses the p26 protein as an an-
tigen [27]. Immune response against gp45 and gp90 envelope
proteins, encoded by env gen [29], is developed in first stage of
infection. Previous studies have reported that antibodies against
capsid p26 protein are produced after glycoproteins antibodies
because gp45 is a transmembrane protein and gp90 an integral
membrane protein, both more exposed from immune system
recognition [30,31]. Despite the rapid and high rate of env gene
mutations, as a viral mechanism for evasion of the immune system
response, conserved regions in the env sequence have been iden-
tified for the diagnostic and production of immunogenic peptides
[24,32,33].
Enzyme-linked immunosorbent assay system reached an
appropriate performance in comparison with AGID, which is the
method accepted for EIA diagnosis by the OIE. The analysis of the
results demonstrates high levels of sensitivity (100%) and speci-
ficity (97.6%), despite the low number of samples evaluated. The
strong points of the ELISA AIE-LAB arising from this assay were
diagnostic sensibility and precocity, which could be due to the
antigen used. False negative results can be the product of low
specificity or sensitivity of diagnostic test, making difficult to
establish a good control program for disease eradication [4].
Different classical methods are been available for antigens prepa-
ration, usually using infected spleen or primary cell culture equine.
These techniques have disadvantages because are expensive,
laborious, and the antigen can be contaminated by another pro-
teins, generating nonspecific results in diagnostic tests [14]. Alter-
native antigen production for serologic diagnosis confers
enhancements in the techniques and consequent improvement of
the programs for EIA eradication [22]. Many authors have reported
the use of envelop synthetic peptides (gp45) as antigen for ELISA
with excellent agreement in comparison with AGID [12,13]. The
calculated ELISA AIE-LAB effectiveness was bigger than 97%, and it
represents a general capacity to detect all true positive and negative
serums correctly. The ELISA AIE-LAB was designed for the screening
of the equine infectious anemia, aiming to bring high sensitivity
and specificity. The concordance degree between both methods
tested was very good (Kappa ¼ 0.9) and demonstrated that this
system is appropriated for the purpose it was designed [14,34].
Control’s serum OD values were correct, and the assay was not
invalidated. No change of coloration, or sedimentation was
observed in the flask of the conjugate. The distilled water used to
dilute the washing solution did not modify the functionality of
Positive Serum
ELISA. The behavior of the general stability of the components and
their functionality after the proper transfer was demonstrated by
X SD CV the OD values of the controls and the result of the precision trial.
1.96 0.05 2.77 In addition, our results agree with those presented recently
1.89 0.11 5.79 where an ELISA using peptides of the gp45 protein as antigen was
evaluated for the diagnosis of serum samples from horse, donkey,

4

zquez Chagoyan et al.
M.C. Rodríguez Domínguez, R. Montes-de-Oca-Jim
enez, J.C. Va
Fig. 2. Principle and method of ELISA AIE-LAB and AGID test.
and mule, with 96.1% concordance, 98.6% sensitivity, and 95.6%
specificity compared with AGID. The sensitivity and specificity of
this ELISA was also > 90% when tested in individual equid species,
indicating the potential of the gp45 antigenebased diagnostic
design, especially in donkeys and mules, which have a demon-
strated tendency of equivocal results in AGID [35].
The ELISA is a technique that offers greater sensitivity and
diagnostic specificity, capable of detecting EIAV-specific antibodies
between 10 and 14 days after infection [35], whereas the AGID test
does not have the sensitivity to detect antibodies during the first
day of infection. Unfortunately, animals with low titers of anti-
bodies can originate false negative results, if only AGID test is used
in diagnostic schedules. This situation is very important due to
enhance risk of widespread of the disease because positive animals
escape the diagnostic filter, allowing the mobilization of infected
animals [4]. Despite AGID test limited capacity due to low-
diagnostic sensitivity and difficult line interpretation by visual
recognition, this technique continues as the established official test
for diagnosis of EIA [3]. Consequently, some countries have been
established the ELISA as the sero-screening official diagnostic tool,
with the confirmation of positive samples by AGID test, before
taking any action with the infected animal [3,5,34]. Surveillance
programs in Mexico establish AGID kit as diagnostic test after OIE
indications. Contrasting with the official stablished immunodiffu-
sion test, ELISA is an immunoenzymatic technique that allows
obtaining results in short periods of time, being more effective to
issue criteria and authorization for the transfer of animals, adding
efficiency to equine production and activities. Furthermore, recent
Table 5
Evaluated parameters of ELISA AIE-LAB relative to commercial AGID IDEXX and AGID
AIE-LABIOFAM.
Parameter Percent (%)
Sensitivity 100
Specificity 97.6
PPV 83.3
PNV 97.67
Efficacy 97.91
Concordance (kappa) 0.9 (very good)
Abbreviations: PPV, predictive positive value; PNV, predictive negative value.
5
Journal of Equine Veterinary Science 98 (2021) 103372
studies have shown that the scientific production dedicated to the
investigation of this virus in Mexico is deficient, despite being one
of the main producing countries of equines [18]. The implementa-
tion of the diagnosis using ELISA systems in Mexico could be an
alternative to expand EIA screening studies, as well as carry out
epidemiologic and prevalence analyzes in higher risk areas because
it is an economical technique with high capacity for processing a
large number of samples in a single round test. Taking to account
the results obtained in the present work, we propose the use of
ELISA EIA-LAB as screening tool for routine diagnosis and confir-
mation of positive samples by AGID the gold standard test.
5. Conclusions
The ELISA AIE-LAB shows good results as a diagnostic system
and could be used in Mexico for the detection of specific antibodies
against the EIAV. Considering the limitations of the AGID test, many
authors share the idea that the OIE diagnostic recommendations for
international trade in equines need to be modified by adding the
ELISA in the routine diagnosis. In addition, the combination of the
ELISA and AGID test as a diagnostic algorithm confers greater
sensitivity and specificity, which could improve the accuracy of EIA
surveillance programs in Mexico.
Acknowledgments
This work was supported by Veterinary Medicine Faculty of
Mexico State Autonomy University, Toluca, Mexico. The authors
thank Dr. Alejandro Rivera from CENASA for providing some part of
serum panel for the evaluation of ELISA AIE-LAB.
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